Polymerase chain reaction (PCR) has rapidly become one of the most widely used techniques in molecular biology. It is a rapid, inexpensive and simple means of producing relatively large numbers of copies of DNA molecules (via enzymatic amplification of a specific nucleic acid sequence of interest) from minute quantities of source material, even when the source nucleic acid is of relatively poor quality.
A standard PCR involves preparation of the sample, the master mix of reagents and the oligonucleotide primers, followed by detection and analysis of the reaction products.
Although any protocol of template nucleic acid preparation is acceptable for PCR purposes, it is often best to use as few steps as possible in the nucleic acid preparation in order to prevent yield reduction and/or accidental contamination with unwanted nucleic acid.
Nucleic acid-based diagnostic procedures in commercial and academic laboratories often require nucleic acid extractions from natural substances. Applications range from forensic DNA-fingerprinting to medical, agricultural and environmental monitoring. It is important that any nucleic acid extraction be free from contamination particularly where concentration of nucleic acid in the initial sample is very low and/or where contamination can lead to incorrect outcomes.
This is particularly the case in forensic and evidential analyses where quantities of starting material may be measured in picograms or less. Contamination of the sample may occur simply as a result of the sample tube being opened to the atmosphere or being touched by a technician.
Because of the ease with which a sample can be contaminated, it is a requirement that reproducible nucleic acid extraction techniques are free from contamination and require protocols directed to minimising such contamination.
Standard nucleic acid extraction techniques are problematic as the sample tube may require opening and shutting at stages throughout the extraction procedure. It would therefore be advantageous to develop a protocol enabling simple, closed-tube reactions minimising the likelihood of contamination.
PCR technology often necessitates lengthy purification procedures. These procedures involved long incubations with proteinases, phenol and chloroform extractions, and finally an ethanolic salt precipitation.
Numerous methods have been described for the preparation of nucleic acid from animal tissue for amplification by PCR. A typical example is described by Hunt, Parks and Limley (1997) Food Chemistry Vol 60: 437-442.
Typically methods for DNA extraction from animal tissue samples (such as meat or bone) contain the following steps:                1) Resuspension of the tissue sample in a buffer containing sodium dodecyl-sulphate (SDS).        2) Homogenisation.        3) Incubation with the enzyme proteinase K for 1-2 hours.        4) Solvent extraction of the sample with phenol.        5) Solvent extraction with a mixture of phenol/chloroform/isoamylalcohol.        6) Solvent extraction with chloroform.        7) Precipitation for a minimum of 1 hour in 3M sodium acetate and 3 volumes of ethanol.        8) Centrifugation of DNA.        9) Washing of the pellet 2 times in ethanol.        10) Air-drying and resuspension of the pellet in buffer.        
Simpler methods are available for the release of DNA from blood. Most commonly, the commercial product Chelex™ is used. These methods provide moderate yields of DNA by simply boiling the sample in the presence of this agent.
However, to remove inhibition of the PCR, forensic scientists routinely pre-wash and centrifuge blood cells to lyse the red cells and remove the haem. This necessitates a further step and source of contamination to the procedure and results in a loss of yield with degraded or environmentally compromised blood samples (typical of crime-scene samples).
The applicant has conducted experiments which show nucleic acid extraction using new proteinases removes this inhibition, thus eliminating the need for this step and resulting in a reduced potential for contamination of samples and an improved yield.
Other standard techniques used in molecular biology may also benefit from simple, closed tube reactions, for example the removal of restriction enzymes and phosphatases that are not heat labile and require time consuming phenol/chloroform extractions, ethanolic salt precipitations and wash steps to purify the sample.
All references, including any patents or patent applications cited in this specification are hereby incorporated by references. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art, in New Zealand or in any other country.
It is acknowledged that the term ‘comprise’ may, under varying jurisdictions, be attributed with either an exclusive or an inclusive meaning. For the purpose of this specification, and unless otherwise noted, the term ‘comprise’ shall have an inclusive meaning—i.e. that it will be taken to mean an inclusion of not only tile listed components it directly references, but also other non-specified components or elements. This rationale will also be used when the term ‘comprised’ or ‘comprising’ is used in relation to one or more steps in a method or process.
It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice.
Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only.